L-arginine is a substrate for nitric oxide synthases (NO synthases) and is a precursor of nitric oxide (NO), a major cell signaling molecule implicated in the regulation of many cellular pathways. The L-arginine: NO pathway has been implicated in the regulation of the cardiovascular, nervous and immune systems. Inhibitors of NO have been shown to increase blood pressure in guinea pigs and rabbits (Aisaka et al Biochem. Biophys. Res. Commun. (1989) 160:-881-886; Rees et al. PNAS (1989) 86:3375-3378), and to induce arteriolar vasoconstriction in humans (Vallance et. al. (1989) Lancet 8670:997-1000). A variety of arginine analogs were identified which modulate the L-arginine:NO pathway (for review see Leiper and Vallance, Cardiovascular Research, 43, 1999, 542-548). These include NG-monomethyl-L-arginine (L-NMMA), NG-, NG-dimethylarginine (ADMA; asymmetric dimethyl arginine) and NGxe2x80x2-, NG-dimethylarginine (SDMA; symmetric dimethylarginine).
In one embodiment, the invention pertains to a method for determining arginine compound levels in body samples of a subject. The method includes contacting a body sample with an arginine sensing substance, and analyzing the resulting mixture. Examples of preferred body samples include, body fluids such as blood, saliva, sweat and urine. In an advantageous embodiment, the body sample is obtained non-invasively. In particular preferred embodiment, the arginine compound level is analyzed through a color change, e.g., a change in optical characteristics or fluorescence, of the arginine compound sensing substance and body fluid mixture. Such determination could determine need for therapy administration or other interventions. Examples of arginine, NG-compounds include L-arginine and derivatives of arginine such as methyl arginine, monomethyl-L-arginine (L-NMMA), NG, NG-dimethylarginine (ADMA; asymmetric dimethyl arginine) and NGxe2x80x2, NG-dimethylarginine (SDMA; symmetric dimethylarginine).
The invention also pertains to a kit suitable for determining arginine compound levels in a subject. Preferably, the kit includes direction for use. In one embodiment of the kit, the arginine sensing substance is embedded in a solid, permeable substrate. In another embodiment the kit includes a vial for mixing an arginine (or arginine compound) sensing substance with a body sample.
The invention also pertains, at least in part, to arginine compound recognizing substances of the formula (I):
G(N)n(C)mxe2x80x83xe2x80x83(I)
wherein
G is a guanidinium recognizing moiety;
N is an ammonium recognizing moiety;
C is a carboxylate recognizing moiety; and
n and m are each independently integers from 0 to 10.
Arginine (Arg) and asymmetric dimethylarginine (ADMA) are present in human bodily fluids, such as serum and urine, and are derived from the catabolism of proteins containing arginine and methylated arginine residues (Cooke, J. P. Arterioscler. Thromb. Vasc. Biol. 2000, 2032-2037). Levels of ADMA is further regulated via metabolic pathways, such as the major one involving the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Bodily production of nitric oxide (NO), the critical modulator of blood flow and blood pressure (Rees, D. D.; et al. PNAS, 1989, 86, 3375-3378), occurs through metabolism of arginine by the specific enzyme nitric oxide synthase (NOS). While arginine is utilized for NO synthesis, endogenous ADMA, on the contrary, downregulates NO production by inhibiting NOS. Abnormal concentrations of ADMA can serve as indications of various disorders, such as renal failure, endothelial dysfunction, and vascular diseases in general (Cooke, J. P. Arterioscler. Thromb. Vasc. Biol. 2000, 2032-2037). Levels of available arginine are very important for NO synthesis in patients with hypercholesterolemia or atherosclerosis. Thus, detection of arginine and ADMA levels in bodily fluids is useful for diagnosis and treatment of these diseases.
The invention pertains, at least in part, to methods and kits for determining levels of arginine compounds in a body sample. In one embodiment, the invention pertains to a diagnostic kit that can detect levels of arginine compounds in body sample. The invention includes methods for determining the appropriate levels of an arginine compound, to administer to a subject who may be suffering from aberrant arginine compound levels due to an arginine compound related disorder.
In one embodiment, the invention pertains to a method for determining arginine compound levels in a body sample. The method includes contacting a body sample with a arginine sensing substance, and analyzing the resulting mixture. Preferably, the arginine sensing substance is a arginine compound recognizing substance.
The term xe2x80x9cbody samplexe2x80x9d includes body fluids and tissues which may potentially contain arginine compounds. The term xe2x80x9cbody samplexe2x80x9d also includes body fluids. The term xe2x80x9cbody fluidsxe2x80x9d includes all fluids obtained from a mammalian body, including, for example, blood, plasma, urine, serum, saliva, sweat, and spinal and brain fluids. In an embodiment, the arginine compound is methyl arginine, L-NMMA, ADMA, SDMA or L-arginine. Furthermore, the body sample may be either processed (e.g., serum, crushed cellular material) or unprocessed.
The term xe2x80x9carginine compoundxe2x80x9d includes L-arginine and derivatives of arginine such as methyl arginine, NG-monomethyl-L-arginine (L-NMMA), NG, NG-dimethylarginine (ADMA; asymmetric dimethyl arginine) and NGxe2x80x2, NG-dimethylarginine (SDMA; symmetric dimethylarginine). Other arginine derivatives which can be identified using the methods and compositions of the invention are also included. Certain arginine compounds are shown in Table 1.
L-NMMA, an arginine compound, has been found to inhibit the cytotoxic effects of activated macrophages and to prevent the release of nitrate and nitrite derived from L-arginine within these cells. After Furchgott""s endothelium-derived relaxing factor was identified as nitric oxide (Palmer et. al., Nature (1987) 327:524-526), it was discovered that L-NMMA inhibited the generation of endolethial NO from L-arginine (Palmer et. al., Nature (1988) 333:664-666). Subsequently, L-NMMA became a tool to probe into the functions of the L-arginine:NO pathway.
L-NMMA is found naturally in cells as an arginine analog. Additionally, asymmetric and symmetric dimethylarginines have been identified. These substituted methyl arginine compounds affect arginine handling and modulate NO synthesis and its regulated pathways. Determining levels of arginine and other arginine compounds (e.g., methylated derivatives) in tissues and body fluids has a predictive and diagnostic value in predisposition or progression of NO associated disorders.
The arginine compounds, ADMA and SDMA, are the major circulating forms of methylarginine in humans. The presence of methylated arginine residues was noted within specialized proteins including myelin basic protein, heat shock proteins, nuclear and nucleolar proteins (Lischwe et. al., J. Biol. Chem. (1985) 260:14304-14310; Lischwe et al. Biochemistry (1985) 22:6025-6028) but their function remains unclear. A series of protein-arginine methyl transferase enzymes have been identified (Paik et. al. J Biol. Chem. (1968) 243:2108-2114; Ghosh et. al., J. Biol. Chem. (1988) 263:19024-19033). Some have wide substrate specificity such as histone and non histone nuclear proteins and others are more selective. These enzymes can generate L-NMMA and SDMA methylated arginines or L-NMMA and ADMA methylated arginines. The production of methylarginine residues is highly regulated and results in the regulation of several signalling pathways. Proteolysis of proteins containing methylarginine residues leads to the release of free methylarginine residues into the cytoplasm (Kakimoto et. al., J. Biol. Chem. (1970) 245:5751-5758).
Initially it was assumed that following proteolysis the released methylarginine are released into the plasma and cleared by kidney without further catabolism. However, in 1987 Sasaoka and co-workers demonstrated the existence of a pathway for the catabolism of ADMA to citrulline and dimethyl amine in rats (Ogawa et. al., Arch Biochem Biophys (1987) 252:526-537). Enzymes such as dimethylarginine dimethylaminohydrolase have been identified (Ogawa et. al., J. Biol. Chem. (1989) 264:10205-10209). Hence both the synthesis and metabolism of methylarginines are highly regulated in normal states.
L-NMMA and ADMA are effective inhibitors of NO and its regulated pathways. SDMA is not an inhibitor of NO and all three methylated arginines enter cells through cationic amino acid transporters known collectively as the y+ transporter which also transports arginine, lysine and ornithine (Bogle et al., Am. J. Physiol. (1995) 269:C750-C765). Such transport mechanism can result in over five-fold concentration of the methylarginines intracellularly as compared to extracellular concentration. The methylarginines interfere with the generation of NO and the transport of arginine and other cationic amino acids.
The language xe2x80x9carginine compound levelxe2x80x9d includes the amount or concentration of arginine compounds in a body sample. In an embodiment, the arginine compound level of the body sample is indicative of the concentration of arginine compounds in the body. Advantageously, the concentration of the arginine compound in the body can be extrapolated from the arginine compound level determined through the methods and kits of the invention. The invention includes methods and kits which detect the presence or absence of a arginine compound concentration over a certain threshold concentration, which may, advantageously, be adjusted based on the optimal or advantageous arginine compound concentrations for a particular situation or a particular patient (e.g., a patient with an NO related disorder or a cardiovascular disorder). For example, the certain threshold concentration of an arginine compound may be individual to a user or to a group of users, e.g., patients with cardiovascular disorders, etc. In another embodiment, the invention includes methods and kits which detect relative or absolute concentrations of arginine compounds in a body sample.
For example, the arginine compound level of ADMA and SDMA has been determined to be about 500 nM-1 xcexcM in plasma of healthy humans. However, the arginine compound level of other arginine compounds (e.g., L-NMMA) has generally been found to be considerably lower. Methylarginines also are found in body fluids, such as urine, in at a concentration of about 60 xcexcmol/24 hours (Macallister et al., Nephrol Dial. Transplant. (1996) 11:2449-2452).
However, aberrant levels of arginine compounds may either be used to either indicate the presence or the potential presence of an arginine compound associated disorder, e.g., such as a NO related disorder or another disorder characterized by concentration or amounts of arginine compounds in a body sample which can be detected using the methods and compositions of the invention. The term xe2x80x9carginine compound associated disordersxe2x80x9d includes disorders or states which are characterized by the presence or absence of arginine compounds such as L-arginine, ADMA, SDMA, and/or L-NMMA. The term xe2x80x9cNO related disordersxe2x80x9d includes disorders which involve NO at some point of the pathway and which can be identified through the use of the methods and compositions of the invention. The term includes disorders which involve, for example, the L-arginine:NO pathway.
One example of an arginine compound associated disorder is renal failure. In renal failure, methylarginine excretion is diminished and both ADMA and SDMA accumulate in the plasma (Macallister et. al., Br. J. Pharmacol. (1996) 119:1533-1540). Ranges of 0.5-10 xcexcM have been reported. The methods, compositions and kits of the invention could be used to identify the aberrant levels of ADMA and/or SDMA to help diagnose a patient""s disorder.
Aberrant levels of arginine compounds in body samples also can be used to identify or help diagnose other arginine compound related disorders. Levels of ADMA and SDMA are higher in patients with renal failure, which falls post dialysis. High levels of ADMA in renal failure might include sodium handling, increased vascular tone and reactivity, enhanced atherogeneses and effects on immune functions. In children with hypertension levels of ADMA are increased and correlate positively with blood pressure and negatively with circulating levels of nitrogen oxides and NO adducts (Goonasekera et. al., J. Hypertension (1997) 15:901-909). Additionally, levels of ADMA were recently shown to correlate with increased presence of hyperlipidaemia in both animals and humans (Bode-Boger et. al., Biochem. Biophys. Res. Commun. (1996) 219:598-603; Boger et. al., Circulation (1998) 98:1842-1847). This finding suggested that ADMA levels might represent a novel risk factor for cardiovascular disease. Therefore, the use of the methods, kits and substances of the invention, e.g., arginine sensing and/or recognizing substances, could be used to readily identify patients at risk or suffering for arginine related disorders, such as cardiovascular disease.
Other arginine compound associated diseases which involve elevated levels of ADMA include Schizophrenia (Das et al., Neurosci Lett. (1996) 215:209-211), H. pylori infection of gastric mucosa (Fandriks et al., Gastroenterology (1997) 113:1570-1575), Alloxan-induced hyperglycaemia (Masuda et al., Br. J. Pharmacol. (1999) 126:211-218), thrombic microangiopathy (Herlitz et al., Scand J. Urol. Nephrol. (1997) 31:477-479), and atherosclerosis (Boger et al., Circulation (1997) 96:1282-1290; Miyazaki et al., Circulation (1999) 99:1141-1146). Therefore, in another embodiment, the methods, compositions and kits of the invention may be used to identify patients at risk or suffering from any one of these disorders or other disorders characterized by aberrant amounts of arginine compounds present in a body fluid.
Very low levels of NO inhibitors represented by the dimethylarginines have a broad spectrum of biological activities. Effects on the cardiovascular system have been described extensively. In human blood vessels L-NMMA at a concentration of 1 xcexcM causes up to 20% inhibition of bradykinin-induced vasodilatation (Macallister Kidney Int. (1994) 45:737-742). In patients with septic shock, infusion of L-NMMA sufficient to increase the circulating levels of L-NMMA to 5 xcexcM are associated with very substantial (over 70%) increases in vascular resistance and more modest (10-15%) increases in arterial blood pressure (Petros et al., Cardiovascular Res. (1994) 28:34-39). ADMA also produces biological effects at low concentrations and circulating concentrations in the order of 10 xcexcM increase blood pressure by about 15% in guinea-pigs (Valiance et. al., Lancet (1992) 339:572-575). Significant effects of methylarginines on blood vessels probably occur at even lower concentrations, since it is clear that L-NMMA can increase systemic vascular resistance and lower cardiac output without producing major effects on arterial pressure. Low doses of L-NMMA (1 mg/kg) decreases renal blood flow and affects sodium handling in humans but blood pressure is not affected. Low levels of NOS inhibitors may produce chronic effects. In cholesterol fed rabbits, doses of NOS inhibitors that do significantly affect arterial blood pressure, markedly enhance neointima formation and early atherogenesis (Cayatte et al., Arterioscler. Thromb. (1994) 14:753-759). These data indicate that minor degrees of inhibition of NOS can lead to significant biological effects that might have implications for long-term homeostasis of the cardiovascular system.
In one embodiment, the arginine compound level is the arginine level, the L-NMMA level, the SDMA level, the ADMA level or a combination thereof.
The term xe2x80x9carginine levelxe2x80x9d refers to the level, amount, or concentration of arginine in the body sample. Similarly, the term xe2x80x9cL-NMMA levelxe2x80x9d refers to the level, amount or concentration of L-NMMA in the body sample. The terms xe2x80x9cSDMA levelxe2x80x9d and xe2x80x9cADMA levelxe2x80x9d refer to the level, concentration, or amount of SDMA or ADMA in a body sample, respectively.
The term xe2x80x9carginine sensing substancexe2x80x9d includes substances which interact with arginine compounds, such that arginine compounds levels in a body sample can be determined. Advantageously, the determination of the arginine compound level is discernible without the use of laboratory equipment. For example, in an advantageous embodiment, the arginine sensing substance interacts with the arginine compound in a body sample such that the arginine compound level can be determined visually, e.g., by a change in color, hue or intensity of the mixture of the body sample and the arginine sensing substance. The term xe2x80x9claboratory equipmentxe2x80x9d includes HPLC, fluorometers, spectrometers (NMR, IR), optical density meters, etc. The term xe2x80x9claboratory equipmentxe2x80x9d does not include charts or other tables which involve visual comparison of a solution to the chart or table, equipment (e.g., refrigerator, freezer, scissors) usually found in a home, or equipment, e.g., a solution vial, dish, or a syringe, which can be reasonably packaged with the kit without prohibitive expense to the user or another.
The term xe2x80x9cinteractxe2x80x9d or xe2x80x9cinteractionsxe2x80x9d include events which allow for the detection of arginine compounds in a sample. In an embodiment, the term includes electrostatic or hydrogen bonding interactions between the arginine compound recognizing substance and the arginine compound in the sample. In a further embodiment, the interactions are specific for a particular arginine compound.
In other embodiments, the determination of arginine compound levels include additional steps, such as, exposing the mixture to radiation of appropriate wavelength to observe fluorescence. Furthermore, additional substances may be used to detect the presence of an interaction between the arginine sensing substance and a arginine compound. Preferably, the arginine sensing substance interacts specifically with L-arginine, L-NMMA, ADMA, SDMA, or another arginine analog which is indicative of an arginine compound associated disorder or a disorder of the L-arginine NO pathway. In one embodiment, the arginine sensing substance comprises a polypeptide, e.g., an antibody or a fragment thereof, which binds to the arginine compounds. In another embodiment, the arginine sensing substance comprises a cage molecule, such as, for example, a fullerene. Advantageously, the arginine sensing substance specifically interacts with a specific arginine compound, for example, L-arginine, L-NMMA, ADMA, or SDMA. For example, a L-arginine sensing substance may interact specifically with L-arginine to indicate the L-arginine level in a body sample. Similarly, an ADMA sensing substance would interact specifically with ADMA to indicate the ADMA level in a body sample. Furthermore, one or more sensing substances may be used in combination to specifically detect several arginine compounds separately. Furthermore, the term xe2x80x9carginine sensing substancesxe2x80x9d includes xe2x80x9carginine compound xe2x80x9crecognizing substances.xe2x80x9d
The term xe2x80x9carginine compound recognizing substancesxe2x80x9d includes substances which specifically interact with arginine compounds. The arginine compound recognizing substances may be specific for certain arginine compounds, e.g., L-arginine (e.g., xe2x80x9cL-arginine recognizing substancesxe2x80x9d), L-NMMA (e.g, xe2x80x9cL-NMMA recognizing substancesxe2x80x9d), ADMA (e.g., xe2x80x9cADMA recognizing substancesxe2x80x9d), or SDMA (e.g., SDMA recognizing substancesxe2x80x9d) or salts or ions thereof. The interaction of arginine compounds with arginine compound recognizing substances can be detected without modification of the arginine or arginine compound, or the production of an enzymatic product. However, deprotonation or protonation of acidic or basic groups of arginine compounds is not considered to be modification of the arginine compounds.
Arginine compound recognizing substances involve specific interactions between the substances and the arginine compounds. The language xe2x80x9cspecifically interactxe2x80x9d or xe2x80x9cspecific interactionsxe2x80x9d is not intended to include general methods of separation and detection, such as chromatographic techniques (e.g., HPLC) which use, for example, molecular weight, charge, or vaporization point to separate molecules with similar physical properties. The language xe2x80x9cspecifically interactxe2x80x9d or xe2x80x9cspecific interactionsxe2x80x9d includes interactions between the arginine compound recognizing substance and the arginine compound which are capable of identifying the arginine compound based on its structural properties on a molecular level, such as the size, location and polarity of chemical moieties of the arginine compound. Furthermore, the term xe2x80x9carginine compound sensing substancesxe2x80x9d includes xe2x80x9carginine compound recognizing substances.xe2x80x9d In a preferred embodiment, the arginine compound recognizing substance specifically interacts with arginine, L-NMMA, ADMA, or SDMA.
Examples of arginine compound recognizing substances include, for example, antibodies which detectably interact with arginine compounds and other organic and organometallic molecules.
In a preferred embodiment, the arginine compound recognizing substance is an organic small molecule. The term xe2x80x9corganic small moleculexe2x80x9d includes organic and organo-metallic molecules. In one embodiment, the organic small molecules of the invention interact with arginine compounds such that the presence or concentration of arginine compounds in a sample can be determined.
In one embodiment, the arginine compound recognizing substance is an organic small molecule which specifically interacts with arginine compounds, such as L-arginine, L-NMMA, ADMA, SDMA, etc. These compounds can be synthesized and designed using the techniques and design strategy of xe2x80x9chost-guestxe2x80x9d chemistry, in which a receptor is designed to the specification of the xe2x80x9cguestxe2x80x9d (e.g., the arginine compound.) Examples of the methods for designing hosts (recognizing substances) can be found in U.S. Pat. Nos. 5,030,728, and 5,128,466.
Over the past 30 years, chemists have designed and synthesized many organic compounds capable of interacting with other organic molecules. These organic compounds are also termed xe2x80x9chostxe2x80x9d compounds or xe2x80x9cartificial receptorsxe2x80x9d by analogy with biological receptors that bind and recognize xe2x80x9cguests.xe2x80x9d xe2x80x9cHost-guestxe2x80x9d or supramolecular chemistry, has numerous biomedical applications, including detection and quantitation of analytes, such as arginine compounds, in biological fluids. In one embodiment, the invention includes xe2x80x9chostxe2x80x9d arginine sensing substances which interact with arginine compounds.
For example, xe2x80x9chost-guestxe2x80x9d supramolecular chemistry has been used to create diketone xe2x80x9chostsxe2x80x9d which interact with urea xe2x80x9cguestsxe2x80x9d (Bell, T. W. et al. J. Am. Chem. Soc. (1988) 110:3673-3674). The strength of this complex was thought to be due to the relative rigidity of the diketone xe2x80x9chostxe2x80x9d prior to interaction with the urea, and the position of the diketone""s hydrogen-bond acceptor nitrogen and oxygen atoms in nearly ideal locations to form strong hydrogen bonds with the arginine compound""s NH stabilizing groups. The principle of preorganization includes both the effect of the conformational organization of the host and the low solvation of the binding site before complexation (Cram, D. J. Angew. Chem. Int. Ed. Engl. (1986) 25:1039-1134). Other series of highly preorganized hydrogen bonding receptors for various guest molecules have been also synthesized and discussed (Bell, T. W. et al. Angew. Chem. Int. Ed. Engl. (1997) 36:1536-1538; Bell, T. W. et al. Angew. Chem. Int. Ed. Engl., (1990) 29:923-925; Beckles, D. L. et al. Tetrahedron (1995) 51:363-376; U.S. Pat. Nos. 5,030,728; and 5,283,333).
Arginine compound recognizing substances, e.g., organic small molecules, designed to interact with arginine compounds and, advantageously, signal this interaction may be used to determination of the arginine compound levels in body samples.
One example of a the hexagonal lattice approach to a water-soluble arginine receptor, the xe2x80x9carginine corkxe2x80x9d is shown in Scheme 1 (Bell et al., Angew. Chem., Int. Ed. Engl., 1999, 38, 2543-2547). Arginine compound recognizing substance (1) interacts with alkylguanidinium ions, the side chain of arginine, in water with formation of a complex, with arginine. The dissociation constant of the complex of 1 with arginine in water was found to be 1.1 mM. Electrostatic attraction between negatively charged carboxylate groups of 1 and the positive charge of the guanidinium ion together with the preorganized network of hydrogen-bond acceptor sites of the receptor make the complex of 1 with guanidinium ion to be highly stable. However, receptor 1 binds any alkylguanidinium compounds and lacks recognition specificity, which is important for selective sensor. 
In another example, a rigid U-shaped guanidinium receptor (2) (Scheme 2) changes its light absorption properties upon complexation to unsubstituted guanidinium ion (Bell et al., Angew. Chem. Int. Ed. Eng. 1990, 29, 923-925). Receptor 2 is restricted by design to bind only to unsubstituted guanidinium ion. 
The invention pertains, at least in part, to a series of arginine compound recognizing substances designed to interact with arginine compounds selectively, e.g., by utilization of complementary electrostatic and preorganized hydrogen-bonding interactions. In one embodiment, the arginine compound recognizing substances of the invention are of the formula (I):
G(N)n(C)mxe2x80x83xe2x80x83(I)
wherein
G is a guanidinium recognizing moiety;
N is an ammonium recognizing moiety;
C is a carboxylate recognizing moiety; and
n and m are each independently integers from 0 to 10.
Generally, arginine has several functional groups, guanidinium, ammonium and carboxylate, which may be targeted for recognition. In an embodiment, the arginine compound recognizing substances comprises a guanidinium recognizing moiety (G), an ammonium recognizing moiety (N), and a carboxylate recognizing moiety (C). The arginine compound recognizing substance may also further comprise linking moieties which connect the guanidinium recognizing moiety, the ammonium recognizing moiety, and the carboxylate recognizing moiety.
In an embodiment, the arginine compound recognizing substances have the general structure shown in Scheme 3. 
The term xe2x80x9cguanidinium recognizing moietyxe2x80x9d (xe2x80x9cGxe2x80x9d) includes moieties which coordinate with arginine compounds. In an embodiment, the moiety interacts with the guanidinium moiety of the arginine compound. Preferably, the guanidinium recognizing moiety detectably coordinates with the arginine compounds at biological concentrations. In an embodiment, the guanidinium coordinating moiety is multicyclic, and may advantageously contain at least one heterocycle, e.g., a nitrogen containing heterocycle, e.g., a pyridyl moiety. In an embodiment, the guanidinium recognizing moiety is hydrogen-bond accepting and/or anionic.
The guanidinium recognizing moiety may be designed such that it specifically recognizes non-methylated guanidinium group of arginine (e.g. a xe2x80x9cnon-methylated guanidinium recognizing moietyxe2x80x9d), the monomethylated guanidinium group of NMMA (e.g, a xe2x80x9cmonomethylated guanidinium recognizing moietyxe2x80x9d), the symmetric dimethylation of the guanidinium group of SDMA (e.g. a xe2x80x9csymmetric dimethylated guanidinium recognizing moietyxe2x80x9d) or the asymmetric guanidinium group of ADMA (e.g., an xe2x80x9casymmetric dimethylated guanidinium recognizing moietyxe2x80x9d, xe2x80x9cQxe2x80x9d). The term xe2x80x9cguanidinium recognizing moietyxe2x80x9d includes each of these recognizing moieties (e.g. non-methylated guanidinium recognizing moiety, monomethylated guanidinium recognizing moiety, and the symmetric and asymmetric guanidinium recognizing moieties.)
Examples of non-methylated guanidinium recognizing moieties for arginine recognizing substances include the following structures shown below. 
Furthermore, other examples of guanidinium recognizing moieties include derivatives and analogs of the guanidinium recognizing moieties shown above. For example, the guanidinium recognizing moieties shown above can be substituted with various functional groups to enhance their ability to perform their function, e.g., detect arginine compound levels. Analogs include, for example, compounds and moieties which are structurally similar but may have substitutions of heteroatoms or other changes which do not prohibit the guanidinium recognizing moiety or the arginine compound recognizing substance from performing its intended function, e.g., determine arginine compound levels in a body sample. In an advantageous embodiment, the analogs or derivatives of the guanidinium recognizing moieties shown above enhance the ability of the arginine compound recognizing substance to perform its intended function.
The term xe2x80x9casymmetric dimethylated guanidinium recognizing moietyxe2x80x9d or xe2x80x9cQxe2x80x9d comprises moieties which are capable of interacting with the dimethylguanidinium group such as, for example, rigid heteroaromatic coordinating moieties, such as those shown below: 
The coordinating moiety can be neutral, anionic, or linked to an anionic group, such as carboxylate or phosphate.
The term xe2x80x9cammonium recognizing moietyxe2x80x9d (xe2x80x9cNxe2x80x9d) includes moieties which interact with the ammonium moiety of the arginine compound, such that arginine compound recognizing substance of the invention is capable of performing its intended function, e.g., detect arginine compounds. In a further embodiment, the ammonium recognizing moiety is a neutral or anionic groups and may contain heteroatoms such as nitrogen and oxygen. Examples of ammonium recognizing moieties include, but are not limited to, carbonyl, amide, hydroxyl, hydroxime, carboxylate, ether, ester, pyridine, pyrimidine, phenolate, phosphate, and combinations thereof. Ammonium recognizing moieties may comprise one or several interlinked groups. Some examples of ammonium recognizing moieties are shown below: 
Analogs and derivatives of the ammonium recognizing moieties mentioned above are also included.
The term xe2x80x9ccarboxylate recognizing moietyxe2x80x9d (xe2x80x9cCxe2x80x9d) includes moieties which are capable of interacting with carboxylate moiety of the arginine compound, such that the arginine compound of the invention is capable of performing its intended function. Examples of carboxylate recognizing moieties include neutral and cationic groups. In a further embodiment, the carboxylate recognizing moiety comprises a cationic group, e.g., a guanidinium or ammonium ion, optionally linked to additional hydrogen-bond donating groups such as amine, amide, hydroxyl, hydroxime or a substituted urea. Examples of carboxylate recognizing moieties include but are not limited to: 
wherein Z is alkyl, alkenyl, alkynyl, hydrogen, acyl, hydrogen, and halogen atoms. Analogs and derivatives of the carboxylate recognizing moieties mentioned above are also included.
The term xe2x80x9clinking moietyxe2x80x9d includes moieties which connect (e.g., through covalent bonds) the guanidinium recognizing moiety or the dimethylguanidinium recognizing moiety, the ammonium recognizing moiety, and the carboxylate recognizing moiety. The linking moiety may be a chain of 1 to 30 atoms, optionally substituted, and may contain rings, heteroatoms, single, double, and triple bonds. Advantageously, the linking moiety allows the arginine compound recognizing substance to perform its intended function, e.g., detect arginine compounds.
The arginine compound recognizing substances comprise any combination and order of moieties G, A and C. The combination of moieties G, A, and C can be arranged in linear or cyclic fashion. Some examples of arginine recognizing substances are shown below: 
wherein X is hydrogen, alkyl, alkenyl, alkynyl, aryl, halogen, a chromophore, or a fluorophore. Analogs and derivatives of the arginine recognizing substances mentioned above are also included.
In one embodiment, the arginine sensing substances and the arginine compound recognizing substances of the invention do not include the compounds described in Bell et al., Angew. Chem., Int. Ed. Engl., 1999, 38, 2543-2547.
In one embodiment, the chromophore may be incorporated into the interaction site of the arginine compound on the arginine compound recognizing substance. In one embodiment, the design of arginine compound recognizing substance which changes its optical properties upon complexation of arginine compound of interest. The inclusion of a chromophore or fluorophore may advantageously enhance communication between interaction of the arginine compound with the arginine recognizing substance (Chemosensors of Ion and Molecule Recognition, J. P. Desvergne, A. Czarnik, Eds., Kluwer:Dordrecht, The Netherlands, 1997, pp.121-132).
In another embodiment, the arginine compound recognizing substance is an xe2x80x9cADMA recognizing substancexe2x80x9d which is capable of detectably interacting with ADMA, preferably, specifically. In an embodiment, the ADMA recognizing substances interact specifically with ADMA, e.g., using complementary electrostatic and/or preorganized hydrogen-bonding interactions. ADMA has several distinct groups, dimethylguanidinium, ammonium and carboxylate, which may be targeted for recognition. In an embodiment, the ADMA recognizing substance is of formula (II):
Q(N)n(C)mxe2x80x83xe2x80x83(II)
wherein
Q is an asymmetric dimethylated guanidinium recognizing moiety;
N is an ammonium recognizing moiety;
C is a carboxylate recognizing moiety, and
n and m are each independently integers from 0 to 10.
In an embodiment, the ADMA recognizing substance comprises at least one asymmetric dimethylated guanidinium recognizing moiety Q, at least one ammonium recognizing moiety N, and at least one carboxylate recognizing moiety C. The ADMA recognizing substance may also further comprise linking moieties which connect the dimethylguanidinium recognizing moiety, ammonium recognizing moiety, and the carboxylate recognizing moiety. In one embodiment, the ADMA recognizing substance is represented by the Q-C-N structure in the scheme shown below: 
The ADMA recognizing molecules consist of any combination and order of moieties Q, A and C. The combination of moieties Q, A, and C can be arranged in linear or cyclic fashion. Some examples of ADMA recognizing substances are shown below: 
wherein X is hydrogen, alkyl, alkenyl, alkynyl, aryl, halogen, chromophore or a fluorophore. Analogs and derivatives of the ADMA recognizing substances mentioned above are also included.
The invention also pertains, at least in part, to the arginine compound recognizing substances described herein per se, as well as kits, packages and other products which comprise the arginine compound recognizing substances described herein.
The term xe2x80x9calkylxe2x80x9d includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
Moreover, the term alkyl includes both xe2x80x9cunsubstituted alkylsxe2x80x9d and xe2x80x9csubstituted alkylsxe2x80x9d, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyls can be further substituted, e.g., with the substituents described above. An xe2x80x9calkylarylxe2x80x9d moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
The term xe2x80x9carylxe2x80x9d includes aryl groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as xe2x80x9caryl heterocyclesxe2x80x9d, xe2x80x9cheteroarylsxe2x80x9d or xe2x80x9cheteroaromaticsxe2x80x9d. The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
The terms xe2x80x9calkenylxe2x80x9d and xe2x80x9calkynylxe2x80x9d include unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively.
Unless the number of carbons is otherwise specified, xe2x80x9clower alkylxe2x80x9d as used herein means an alkyl group, as defined above, but having from one to five carbon atoms in its backbone structure. Likewise, xe2x80x9clower alkenylxe2x80x9d and xe2x80x9clower alkynylxe2x80x9d have similar chain lengths.
The terms xe2x80x9calkoxyalkylxe2x80x9d, xe2x80x9cpolyaminoalkylxe2x80x9d and xe2x80x9cthioalkoxyalkylxe2x80x9d include alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.
The terms xe2x80x9cpolycyclylxe2x80x9d or xe2x80x9cpolycyclic radicalxe2x80x9d refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are xe2x80x9cfused ringsxe2x80x9d. Rings that are joined through non-adjacent atoms are termed xe2x80x9cbridgedxe2x80x9d rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term xe2x80x9cheteroatomxe2x80x9d as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
The term xe2x80x9csubstitutedxe2x80x9d includes substituents mentioned above, which include halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
It will be noted that the structure of some of the compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis.
In one embodiment, the levels of the arginine compound can be directly analyzed visually, e.g., by a change in color of the arginine sensing substance and arginine compound mixture.
In one embodiment of the invention, the color, intensity or hue of the arginine sensing substance or a product thereof may be calibrated to indicate a range of arginine compound levels (e.g., the intensity of the color of the arginine sensing substance may intensify as the arginine compound level in the sample is increased; the color or hue of the arginine sensing substance may change as the arginine compound level is decreased.) In a further embodiment, the resulting mixture is analyzed by comparing the color, hue or intensity of the resulting mixture with a calibrated scale, which indicates arginine compound level in the body fluid, or, preferably, in the body. In a further embodiment, the intensity of the color can be determined quantitatively, for example, by measuring changes in the optical density of a solution or by measuring the fluorescence emission.
The term xe2x80x9ccolorxe2x80x9d includes changes in the absorbance or emission radiation in the ultraviolet, visible, or infrared spectrum. Advantageously, the change in color is a change in the visible color of the arginine sensing substance. Alternatively, the change in color could be a change in the wavelength of fluorescence. Furthermore, the level of fluorescence, color or optical change may be quantified, using known spectroscopic (e.g., fluorimetric, colorimetric) techniques.
Examples of arginine compound recognizing substances include molecules capable of specifically interacting with arginine compounds with potentially useful changes in color, light absorption intensity or wavelength, or fluorescence emission intensity or wavelength. Such optical effects can be produced, for example, by rearrangement, transprotonation, ionization, deionization, conformational change, polarization, solvation change or electronic interaction between the arginine compound recognizing substance and the arginine compound. The arginine compound recognizing substances can be designed in a manner similar to that used to design other compounds which are known to generally interact with guanidinium compounds (Bell, T. W. et al. Angew. Chem. Int. Ed., (1999) 38, 2543-2548).
In one embodiment, arginine compound recognizing substances of the invention can be designed advantageously to produce an optical signal, in addition to binding a arginine compound of interest with high affinity. This signal can be, for example, a change in light absorption or emission resulting from a structural change of the arginine compound recognizing substance, electronic polarization of the arginine compound recognizing substance, or other electronic interaction between the compound of interest and the arginine compound recognizing substance.
In a further embodiment, the interaction between the arginine sensing substance and the arginine compound can be detected through the use of fluorescence emission. Quenching or enhancement of emission intensity can result from energetically undemanding processes, such as electronic interaction between arginine sensing substance and the arginine compound of interest or changes in solvation of either substance upon complexation.
In a further embodiment, the method also comprises the step of administering a therapeutically effective amount of a arginine compound to increase arginine compound levels in a subject from which the body fluid sample was taken.
The term xe2x80x9cadministeringxe2x80x9d includes routes of administration which allow the arginine compound to perform its intended function. Examples of routes of administration which can be used include parental injection (e.g., subcutaneous, intravenous, and intramuscular), intraperitoneal injection, oral, inhalation, and transdermal. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the arginine compound can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function. The arginine compound can be administered alone or with a pharmaceutically acceptable carrier. Further, the arginine compound can be administered as a mixture of arginine compounds, which also can be coadministered with a pharmaceutically acceptable carrier. Preferably the arginine compounds are administered orally.
The phrase xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d includes pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it can performs its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer""s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
The language xe2x80x9ctherapeutically effective amountxe2x80x9d includes the amount of the arginine compound sufficient to prevent onset of diseases or significantly reduce progression of such diseases in the subject being treated. A therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the severity of the symptoms to be treated and the activity of the specific analog selected if an analog is being used. Further, the effective amounts of the arginine compound may vary according to the age, sex and weight of the subject being treated. Thus, a therapeutically effective amount of the arginine compound can be determined by one of ordinary skill in the art employing such factors as described above using no more than routine experimentation in clinical management. This therapeutic amount will be linked to levels of arginine compound detected in the assay kit proposed in this invention.
In another embodiment, the invention features a portable kit for determining arginine compound levels in a body fluid. In one embodiment, the kit comprises a arginine sensing substance and instructions for use. The kit may also include a container, vials for the bodily fluids, solvents, and arginine compounds in therapeutically effective amounts.
Although methods for determining arginine levels are currently available, generally these methods are not suitable for use in a kit, because they depend the extensive use of laboratory equipment.
In a preferred embodiment, the arginine sensing substance is associated with a solid support, e.g., embedded in a carrier matrix. Advantageously, the carrier matrix is insoluble in water and other physiological fluids. Examples of carrier matrices include: paper, sponge materials, cellulose, wood, woven and nonwoven fabrics, glass fiber, polymeric films, preformed and microporous membranes, synthetic and modified naturally-occurring polymers, or hydrophilic inorganic powders.
In a further embodiment, the solid support is a arginine compound sensing substance embedded test strip. The test strip may include a support strip, or handle, normally constructed from a hydrophobic plastic, and a reagent test region, containing a bibulous or a nonbibulous carrier matrix incorporating the arginine sensing substance. In one embodiment, the carrier matrix is an absorbent material that allows the body fluid to move, in response to capillary forces, through the carrier matrix to contact the arginine sensing substance and produce a detectable or measurable color transition. In the assay of a whole blood sample, the carrier matrix generally is not permeable to the cellular material. Therefore, the highly-colored cells can be wiped or blotted from the test pad and not interfere with or mask the assay for the arginine compound. Furthermore, if the carrier matrix is permeable to the cellular material, persons of ordinary skill in the art are aware of techniques and devices to separate the cellular material from the test sample to eliminate the interfering affects of the cellular material.
The carrier matrix can be any substance capable of incorporating the arginine sensing substances, as long as the carrier matrix is substantially inert, and is porous or absorbent relative to the soluble components of the liquid test sample. The expression xe2x80x9ccarrier matrixxe2x80x9d refers to either bibulous or nonbibulous matrices that are insoluble in water and other physiological fluids and maintain their structural integrity when exposed to water and other physiological fluids. Suitable bibulous matrices include filter paper, sponge materials, cellulose, wood, woven and nonwoven fabrics and the like. Nonbibulous matrices include glass fiber, polymeric films, and preformed or microporous membranes. Other suitable carrier matrices include hydrophilic inorganic powders, such as silica gel, alumina, diatomaceous earth and the like; argillaceous substances; cloth; hydrophilic natural polymeric materials, particularly cellulose material, like celulosic beads, and especially fiber-containing papers such as filter paper or chromatographic paper; synthetic or modified naturally-occurring polymers, such as crosslinked gelatin, cellulose acetate, polyvinyl chloride, polyacrylamide, cellulose, polyvinyl alcohol, polysulfones, polyesters, polyacrylates, polyurethanes, crosslinked dextran, agarose, and other such crosslinked and noncrosslinked water-insoluble hydrophilic polymers. The carrier matrix can be of different chemical compositions or a mixture of chemical compositions. The matrix also can vary in regards to smoothness and roughness combined with hardness and softness.
The contents of all references and published patents and patent applications cited throughout the application are hereby incorporated by reference.